Microtubules are a fundamental component of the cytoskeleton of eukaryotic cells and are associated with virtually any activity of a cell that involves movement (1). They are part of the mitotic spindle in mammalian cells, and their proper organization is essential for normal cell processes. Although microtubules perform heterogeneous tasks in cells, their basic structure is uniform. The core of the microtubule is entirely composed of tubulin, a 100 kDa heterodimer that assembles to form dynamic cylindrical structures (2).
The carboxy-terminal 15-20 amino acids of each tubulin subunit are the primary locus of sequence heterogeneity in an otherwise highly conserved protein. Tubulin is subject to extensive posttranslational modifications, including acetylation, polyglutamylation, polyglycylation, phosphorylation, tyrosination, and palmitoylation (for recent reviews, see refs 3 and 4). With the exception of acetylation, all of these posttranslational modifications take place in the carboxy-terminal peptides of each subunit. Little is known about the structure of these peptides; they are not observable in the electron or X-ray diffraction structures of tubulin (5, 6). These peptides contain an abundance of glutamic acid residues and so are highly negatively charged at physiological pH. Molecular modeling supports the earlier hypothesis that the carboxy termini extend into solution perpendicularly to the microtubule central axis (7), but there are no experimental data that directly address this question.
A posttranslational event that is unique to tubulin is removal and replacement of the C-terminal tyrosine of α-tubulin (8). In this process, the genetically encoded tyrosine is cleaved by an unknown carboxypeptidase and replaced by the enzyme tubulin tyrosine ligase (TTL). TTL has been isolated from brain tissue, and the human version has been cloned and expressed, but the carboxypeptidase(s) involved in the detyrosination reaction has not yet been identified (9, 10). In vivo, microtubules containing detyrosinated tubulin (Glutubulin) are more stable than those containing tyrosinated tubulin (Tyr-tubulin) (11). Detyrosination does not affect the dynamic or drug-binding properties of purified tubulin (12), and detyrosination of tubulin does not stabilize microtubules in vitro (13); thus, the presence or absence of the α-tubulin carboxy-terminal tyrosine affects the association of nontubulin proteins with cellular microtubules rather than their intrinsic dynamicity. Although the purpose of the enzymatic cycle is not well understood, it is essential for the life of the cell. There is clear evidence that the tyrosination/detyrosination cycle is critical for neuronal organization and may influence tumorigenesis and tumor invasion. For example, TTL null mice undergo normal embryonic development but die shortly after birth (11). Poor patient prognosis has been correlated with elevated levels of Glu-tubulin in breast and prostate tumors and in neuroblastomas (14-16).
Tubulin, the major component of microtubules, undergoes a posttranslational modification that is unique to this protein. The carboxy terminal amino acid on just one of the highly homologous subunits of the tubulin heterodimer is hydrolyzed and replaced by a cycle of two specific enzymes. Tubulin tyrosine carboxypeptidase (TTCP) removes the genetically encoded C-terminal tyrosine from polymerized tubulin, exposing a glutamic acid residue. Tubulin tyrosine ligase (TTL) catalyzes peptide bond formation between this glutamic acid and tyrosine, and this enzyme acts preferentially on tubulin in its depolymerized state. TTL has been isolated and cloned, but nothing is known about the sequence and structure of TTCP. This post-translational modification cycle is critical for neuronal network organization and its disruption can also affect tumorigenesis. TTL is downregulated in some aggressive cancers; thus, molecules that promote tubulin tyrosination—perhaps TTCP inhibitors—may be useful chemotherapeutic agents for these cancers, which currently have poor prognosis.
Previously, in vitro use of purified TTL, tubulin and formyltyrosine was published in 2010, and showed that the reaction between the labeled tubulin and a novel hydrazine fluorophore can take place in a live cell. Abhijit Banerjee, Timothy D. Panosian, Kamalika Mukherjee, Rudravajhala Ravindra, Susannah Gal, Dan L. Sackett, and Susan Bane, “Site-Specific Orthogonal Labeling of the Carboxy Terminus of α-Tubulin”, ACS Chemical Biology. Vol. 5 No. 8, pp 777-785 (2010), expressly incorporated herein by reference.
American Society for Cell Biology December 2012 meeting Poster #268 described in detail a cytostatic effect of formyltyrosine on cell growth. American Society for Cell Biology December 2012 meeting Poster #285 showed that use of formyltyrosine followed by a fluorescent dye results in fluorescently labeled microtubules that can be seen by fluorescence microscopy. Poster #2540 entitled “Labeling Tubulin with Fluorescent Probes in Live Cells” discusses 3fY tagging of tubulin followed by fluorescent labeling of live cells.
The only other method to fluorescently label microtubules in live cells (without genetic manipulation such as Green Fluorescent Protein) is to use fluorescent Taxol. Invitrogen sells two cell-permeable versions of this molecule. However, Taxol greatly affects the microtubules (increased stable polymer). The cells with fluorescently labeled tubulin using the present method are healthy for at least 24 hours after the labeling procedure.
The labeling reaction involves a chemical reaction that occurs between the labeled tubulin and the fluorophore (hydrazine, hydrazide or aromatic aldehyde). This reaction is unexpectedly very fast in PC3 cells. It is well known that the optimal pH for these reactions is around 4, and that the rate of the coupling reaction decreases dramatically above this pH. For this reason, most recent reactions are performed using a catalyst, generally 100 mM aniline. However, this was found to be unsatisfactory for tubulin, and 4-aminophenylalanine may be used as an alternative. This is detailed in Adam R. Blanden, Kamalika Mukherjee, Ozlem Dilek, Maura Loew, and Susan L. Bane, “4-Aminophenylalanine as a Biocompatible Nucleophilic Catalyst for Hydrazone Ligations at Low Temperature and Neutral pH”, Bioconjugate Chem. 2011, 22, 1954-1961, expressly incorporated herein by reference.
It is also well known that sterics and electronic properties of the electrophile and nucleophile are critical to the kinetics of the covalent bond formation as well as the stability of the resulting bond. Jeet Kalia. Ronald T. Raines, “Advances in Bioconjugation”, Curr Org Chem. 2010 January; 14(2): 138-147, expressly incorporated herein by reference. Thus, reactions performed with this system in biological systems invariably use an aromatic amine catalyst or a low pH and typically multiple hours of reaction time. Based on the chemistry of the functional groups, the reactions should take a long time and/or a catalyst and require large excess of fluorophore. See, e.g., Josep Rayo, Neri Amara, Pnina Krief, and Michael M. Meijler, “Live Cell Labeling of Native Intracellular Bacterial Receptors Using Aniline-Catalyzed Oxime Ligation”, J. Am. Chem. Soc. 2011, 133, 7469-7475, expressly incorporated herein by reference. In contrast, labeling occurs within minutes when the fluorophore is added to the cells. Further, the present technology does not use such a large excess of ligand, so background is not a significant issue. Contrast with, Zhiwen Zhang, Brian A. C. Smith, Lei Wang, Ansgar Brock, Charles Cho, and Peter G. Schultz, “A New Strategy for the Site-Specific Modification of Proteins in Vivo”, Biochemistry 2003, 42, 6735-6746, expressly incorporated herein by reference. A final dye concentration of 1 mM was employed, with extensive washing following an overnight staining, and works well for staining membrane bound proteins but results in a high background staining for cytosolic proteins.
In addition to tyrosine ligation, tubulin also undergoes posttranslational polyglutamylation of some of the glutamic acid residues in the carboxy terminus of both alpha and beta subunits. See, Carsten Janke and Jeannette Chloe Bulinski, “Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions”, Molecular Cell Biology, Vo. 12 2011 1773-1786; Edde, B; Rossier, J; Lecaer, J P; et al., “Posttranslational Glutamylation Of Alpha-Tubulin”, Science Vol. 247 No. 4938 Pages 83-85 (1990), each of which is expressly incorporated herein by reference. The enzymes that do this are like tubulin tyrosine ligase. Janke, C; Rogowski, K; Wloga, D; et al., “Tubulin polyglutamylase enzymes are members of the TTL domain protein family”, Science Vol. 308 No. 5729 Pages: 1758-1762 2005. Tubulin tyrosine ligase accepts as a substrate a tyrosine derivative in which the carboxylic acid is a hydrazide.
Tubulin polyglutamylases are therefore possible targets for glutamic acid hydrazides, e.g., alpha-hydrazide.
Other proteins undergo glutamylation. It may be possible to probe for this with a hydrazide derivative of glutamic acid. See, J. van Dijk, J. Miro, J.-M. Strub, B. Lacroix, A. van Dorsselaer, B. Eddé, C. Janke, “Polyglutamylation Is a Post-translational Modification with a Broad Range of Substrates”, J. Biol. Chem., 283 (2008), pp. 3915-3922. Besides tubulins, nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. However, using a proteomic approach, a large number of putative substrates for polyglutamylation in HeLa cells were identified, which serve as in vitro substrates for two polyglutamylases, TTLL4 and TTLL5. Tubulin in cilia also undergoes polyglycylation. See, Ikegami, Koji; Setou, Mitsutoshi, “Unique Post-Translational Modifications in Specialized Microtubule Architecture”, Cell Structure and Function, Vol. 35 No. 1, Pages: 15-22 (2010); C. Regnard, E. Desbruyeres, J. C. Huet, C. Beauvallet, J. C. Pernollet, B. Eddé, “Polyglutamylation of nucleosome assembly proteins”, J. Biol. Chem., 275 (2000), pp. 15969-15976, each of which is expressly incorporated herein by reference; this may be probed by hydrazide.
Otherwise unknown posttranslational modification with amino acids may be found. See, George A. Khoury, Richard C. Baliban, & Christodoulos A. Floudas, “Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database”, Scientific Reports 1, 90 doi:10.1038/srep00090 (2011). See also Woodsmith J, Kamburov A, Stelzl U, “Dual Coordination of Post Translational Modifications in Human Protein Networks”. PLoS Comput Biol 9(3): e1002933. doi:10.1371/journal.pcbi.1002933 (2013), each of which is expressly incorporated herein by reference.
Ikeda-Boku A, Ohno S, Hibino Y, Yokogawa T, Hayashi N, Nishikawa K, “A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli”, J Biochem. 2013 March; 153(3):317-26. doi: 10.1093/jb/mvs153. Epub 2013 Jan. 10, expressly incorporated herein by reference, discloses an example of the “unnatural amino acid” technology that could be used to put unnatural amino acids such as 3-formyltyrosine or 3-acetyltyrosine into any protein.
Typically, the reaction between the nucleophile and electrophile would be expected to be slow. Jun Y. Axupa, Krishna M. Bajjuric, Melissa Ritland, Benjamin M. Hutchins, Chan Hyuk Kim, Stephanie A. Kazane, Rajkumar Halder, Jane S. Forsyth, Antonio F. Santidrian, Karin Stafin, Yingchun Lu, Hon Tran, Aaron J. Seller, Sandra L. Biroc, Aga Szydlik, Jason K. Pinkstaff, Feng Tian, Subhash C. Sinha, Brunhilde Felding-Habermann, Vaughn V. Smider, and Peter G. Schultza, “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids”, PNAS, Oct. 2, 2012, vol. 109, no. 40, pp. 16101-16106, expressly incorporated herein by reference, teach that the coupling reaction takes 4 days.
Kazane S A, Sok D, Cho E H, Uson M L, Kuhn P, Schultz P G, Smider V V, “Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR”, Proc Natl Acad Sci USA. 2012 Mar. 6; 109(10):3731-6. doi: 10.1073/pnas.1120682109. Epub 2012 Feb. 15, expressly incorporated herein by reference, teaches coupling conditions which employ a pH of 4.5 and 100 mM methoxyaniline.
Rayo J, Amara N, Krief P, Meijler M M, “Live cell labeling of native intracellular bacterial receptors using aniline-catalyzed oxime ligation”, J Am Chem Soc. 2011 May 18; 133(19):7469-75. doi: 10.1021/ja200455d. Epub 2011 Apr. 22, expressly incorporated herein by reference, employs an aniline catalyzed oxime formation in a live bacterial cell system. In this system, aniline (1 mM) catalyzes the reaction between aldehydes and oxyamines at neutral pH, resulting in oxime formation with good reaction rates, with labeling occurring over a period of 12 hours.
Rashidian M, Song J M, Pricer R E, Distefano M D, “Chemoenzymatic reversible immobilization and labeling of proteins without prior purification”, J Am Chem Soc. 2012 May 23; 134(20):8455-67. doi: 10.1021/ja211308s. Epub 2012 May 8, expressly incorporated herein by reference, provides a reaction which was initiated by adding 100 mM aniline and allowed to proceed for 1 h at room temperature.
Yao J Z, Uttamapinant C, Poloukhtine A, Baskin J M, Codelli J A, Sletten E M, Bertozzi C R, Popik V V, Ting A Y, “Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition”, J Am Chem Soc. 2012 Feb. 29; 134(8):3720-8. doi: 10.1021/ja208090p. Epub 2012 Feb. 14, expressly incorporated herein by reference, employs a 10 minute incubation followed by an extensive 2 hours of washing to remove excess fluorophore prior to imaging.
Georg C Rudolf, Wolfgang Heydenreuter and Stephan A Sieber, “Chemical proteomics: ligation and cleavage of protein modifications”, Current Opinion in Chemical Biology 2013, 17:110-117, expressly incorporated herein by reference, discusses the attachment of molecular modifications onto proteins for various applications. However, no hydrazine/aldehyde couplings are mentioned in this review.
See also, Paresh Agarwala, Joep van der Weijdena, Ellen M. Slettena, David Rabukab, and Carolyn R. Bertozzi, “A Pictet-Spengler ligation for protein chemical modification”, PNAS Jan. 2, 2013, vol. 110, no. 1, pp. 46-51 www.pnas.org/cgi/doi/10.1073/pnas.1213186110; See, Kathrin Lang, Lloyd Davis, Jessica Torres-Kolbus, Chungjung Chou, Alexander Deiters, and Jason W. Chin, “Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction”, Nature Chemistry Vol. 4 (2012) pp. 298-304; Arshad Desai and Timothy J. Mitchison, “Microtubule Polymerization Dynamics”, Annu. Rev. Cell Dev. Biol. 1997. 13:83-117; Goodson, Holly V, “Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP—tubulin and characterization of clones”, Cold Spring Harbor protocols, Vol. 2010 Issue 9, Pages: pdb.prot5480; Sohye Jang, Kalme Sachin, Hui-jeong Lee, Dong Wook Kim, and Hyun Soo Lee, “Development of a Simple Method for Protein Conjugation by Copper-Free Click Reaction and Its Application to Antibody-Free Western Blot Analysis”, Bioconjugate Chem. 2012, 23, 2256-2261; and Moritz J. Schmidt and Daniel Summerer, “A need for speed; genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells”, Chembiochem; a European journal of chemical biology, Vol. 13 Issue 11 (2012), pp. 1553-1557, each of which is expressly incorporated herein by reference.
Methods available for microtubule labeling and associated disadvantages
MethodsDisadvantage1 Recombinant GFP is a 27 kD protein (vs tubulin, protein expression which is 2 50 kD monomers)(GFP/RFP tubulin)Only 3% of tubulin is labeled-cells not viable with higher labelingRequires genetic manipulation2 Use of drugs conjugated Interferes with microtubule with probesdynamicsExample:Example:BODIPY FL Vinblastine Vinblastine inhibits tubulin(Invitrogen)polymerizationVinblastine 4′-anthranilate Taxol promotes tubulin (Invitrogen)polymerizationTubulinTracker Green reagent (Invitrogen)Oregon Green 488 paclitaxel bis-acetate (Invitrogen)3 MicroinjectionSingle cell manipulationTedious process4 Other probes for tubulinNon-specific—useful with Example:pure protein onlyDCVJ (4-(dicyanovinyl)Example:julolidine(Invitrogen)DCVJ (4-(dicyanovinyl)julolidine: Alsobinds to bovine brain calmodulin5 GTP analog modified with Non-specific—useful with TAMRA, Cy3, or Cy5 pure protein only(BioTechniques 50:43-48Jul. 2011)